Our genome wide association study (GWAS) for familial Parkinson's disease (PD) was the first to identify the cyclin G-associated kinase (GAK) gene as associated with PD risk. Meta-PD GWAS analyses have now demonstrated an unequivocal genome-wide effect (at least P = 4.8 x 10-15) for the rs1564282 SNP in the GAK gene on increased PD risk. Notably, the effect appears to be particularly strong in familial PD. Recently, we published that GAK is associated with 1-synuclein toxicity. Microarray expression analysis of post-mortem frontal cortex from PD and control brains found a significant association of rs1564282 with increased 1- synuclein expression, which has implications for disease pathogenesis. Further, knockdown of GAK significantly increases 1-synuclein toxicity in neuronal cell models of PD. GAK plays a critical role in endocytosis through its interaction with cathepsin-D (CTSD), which is the main lysosomal enzyme involved in 1-synuclein degradation. Taken together, these studies implicate a novel role for GAK in PD pathogenesis. Since kinases, such as GAK, are attractive targets for therapeutic intervention, resolving the responsible functional variants in the GAK gene region and their effects on GAK's expression and function promise important advancements for PD research and therapeutics. This application proposes to identify the responsible mutations, and to further evaluate their role in the implicated endocytic pathway and in the pathogenesis of PD. We propose a definitive series of DNA sequencing, RNA sequencing, alternative splicing, gene expression, and clinical risk profiling studies which have important translational implications. Specifically, Aim 1 will identify functional sequence variants in the GAK region by resequencing 225 Kb in 480 familial PD cases and 96 controls from our GenePD familial PD cohort, in which the gene was identified, thus greatly enhancing the likelihood for successful discovery of functional variants. Aim 2 proposes to perform RNA-sequencing in a large well characterized series of 34 PD and 29 control brains, for which extensive SNP genotyping and microarray data are already available. The RNA-Seq data will be used to study coding sequence and expression of GAK and related genes in the endocytic pathway. RNA sequencing will also allow us to examine all the GWAS implicated PD genes (e.g. SNCA, MAPT, BST1 etc.) and to perform transcriptome-wide comparison of cases and controls. Finally, Aim 3 explores the characteristics of PD cases carrying the variants and mutations found in Aims 1 and 2 and utilizes the genotyping of identified sequence variants in gene-gene and gene- environment interaction and genetic risk profiling studies. Genetic risk profiling studies have important translational implications in risk prediction and diagnostics for PD as well as for interpretation of therapeutic response in clinical trials for PD. These PD investigators are uniquely experienced with the GAK gene and region and have already in hand all the samples needed to carry out these definitive studies.